肝细胞生长因子促进骨形态发生蛋白9诱导小鼠骨髓间充质干细胞C3H10成骨分化的研究  

HGF promotes osteogenic differentiation in mesenchymal stem cells induced by bone morphogenetic protein 9

作　　者：惠慧[1] 罗聪[1] 毕杨[1] 

机构地区：[1]重庆医科大学附属儿童医院儿科研究所干细胞实验室儿童发育疾病研究教育部重点实验室儿科学重庆市重点实验室重庆市儿童发育重大疾病诊治与预防国际科技合作基地,重庆400014

出　　处：《第三军医大学学报》2014年第23期2380-2384,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金青年科学基金(81100309)~~

摘　　要：目的探讨肝细胞生长因子(hepatocyte growth factor,HGF)联合骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)诱导小鼠骨髓间充质干细胞(C3H10)成骨分化的影响,并探讨其可能的分子机制。方法用重组腺病毒介导BMP9在C3H10细胞中过表达,同时联用20 ng/m L肝细胞生长因子HGF诱导C3H10细胞,Real-time PCR检测C3H10成骨分化相关基因骨钙蛋白(osteocalcin,OC)及骨桥蛋白(osteopontin,OPN)基因mRNA水平的表达变化;Western blot检测OC、OPN的蛋白表达水平;ALP染色及ALP读数分析C3H10细胞碱性磷酸酶(alkaline phosphatase,ALP)活性的变化;茜素红S染色检测细胞的钙盐沉积;Real-time PCR检测经典信号通路Smad1/5/8在此诱导过程中的表达水平。结果 BMP9单独作用能诱导C3H10细胞向成骨方向分化,并增强细胞ALP活性,单独HGF有较弱的成骨诱导作用,但与BMP9联用可显著增强C3H10细胞成骨特异性标志物OC与OPN的mRNA及蛋白表达水平(P<0.05),促进ALP活性,及晚期的钙盐沉积;HGF能刺激Smad1/5/8信号通路mRNA表达水平显著增高(P<0.05)。结论肝细胞生长因子可显著增强BMP9诱导C3H10细胞成骨分化的作用,可能是通过激活Smad经典信号通路来实现的。Objective To determine the effect of hepatocyte growth factor（ HGF） combined with bone morphogenetic protein 9（ BMP9） on the induced osteogenic differentiation of mesenchymal stem cell（ MSC） line C3H10 and investigate the possible underlying mechanism. Methods C3H10 cells were treated by recombinant adenovirus expressing Ad-BMP9 infection and HGF（ 20 ng / m L） induction. The expression of osteocalcin（ OC） and osteopontin（ OPN） at mRNA and protein levels were determined by real-time PCR and Western blot analysis respectively. The activity of alkaline phosphatase（ ALP） was detected by ALP staining and activity assay. Calcium deposition was detected by Alizarin Red S staining. At last,the mRNA expression of Smad1 /5 /8 was analyzed by real-time PCR. Results BMP9 alone induced the osteogenic differentiation of C3H10 cells and increased the ALP activity. HGF alone slightly induced osteogenic differentiation,while significantly promoted the BMP9-induced OC and OPN expression（ P 〈 0. 05）,ALP activity,and calcium deposition. Furthermore,HGF induction resulted in significant increase in the transcription levels of Smad1 /5 /8（ P 〈 0. 05）. Conclusion HGF synergistically promotes the effect of BMP9 on induction of osteogenic differentiation of C3H10 cells,and this effect maybe due to the activation of Smad signaling pathways.

关 键 词：骨形态发生蛋白9 肝细胞生长因子 骨髓间充质干细胞 成骨分化 

分 类 号：R322.47[医药卫生—人体解剖和组织胚胎学] R322.71[医药卫生—基础医学]